The relationship between elastin cross linking and alveolar wall rupture in human pulmonary emphysema.

To better define the role of mechanical forces in pulmonary emphysema, we employed methods recently developed in our laboratory to identify microscopic level relationships between airspace size and elastin-specific desmosine and isodesmosine (DID) cross links in normal and emphysematous human lungs. Free DID in wet tissue (a biomarker for elastin degradation) and total DID in formalin-fixed, paraffin-embedded (FFPE) tissue sections were measured using liquid chromatography-tandem mass spectrometry and correlated with alveolar diameter, as determined by the mean linear intercept (MLI) method. There was a positive correlation between free lung DID and MLI (P < 0.0001) in formalin-fixed lungs, and elastin breakdown was greatly accelerated when airspace diameter exceeded 400 µm. In FFPE tissue, DID density was markedly increased beyond 300 µm (P < 0.0001) and leveled off around 400 µm. Elastic fiber surface area similarly peaked at around 400 µm, but to a much lesser extent than DID density, indicating that elastin cross linking is markedly increased in response to early changes in airspace size. These findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.NEW & NOTEWORTHY The current findings support the hypothesis that airspace enlargement is an emergent phenomenon in which initial proliferation of DID cross links to counteract alveolar wall distention is followed by a phase transition involving rapid acceleration of elastin breakdown, alveolar wall rupture, and progression to an active disease state that is less amenable to therapeutic intervention.

The Proinflammatory Activity of Structurally Altered Elastic Fibers.

The mechanisms responsible for the increased loss of pulmonary function following acute lung inflammation in chronic obstructive pulmonary disease remain poorly understood. To investigate this process, our laboratory developed a hamster model that uses a single intratracheal instillation of LPS to superimpose an inflammatory response on lungs treated with intratracheal elastase 1 week earlier. Parameters measured at 2 days after LPS included total leukocyte content and percent neutrophils in BAL fluid (BALF), and BALF levels of both total and peptide-free elastin-specific crosslinks, desmosine and isodesmosine (DID). Airspace enlargement, measured by the mean linear intercept method, and relative interstitial elastic fiber surface area were determined at 1 week after LPS. Compared with animals only treated with elastase, those receiving elastase/LPS showed statistically significant increases in mean linear intercept (156.2 vs. 85.5 µm), BALF leukocytes (187 vs. 37.3 × 104 cells), neutrophils (39% vs. 3.4%), and free DID (182% vs. 97% of controls), which exceeded the sum of the individual effects of the two agents. Despite increased elastin breakdown, the elastase/LPS group had significantly greater elastic fiber surface area than controls (49% vs. 26%) owing to fragmentation and splaying of the fibers. Additional experiments showed that the combination of elastin peptides and LPS significantly enhanced their separate effects on BALF neutrophils and BALF DID in vivo and leukocyte chemotaxis in vitro. The results suggest that structural changes in elastic fibers have proinflammatory activity and may contribute to the decline in pulmonary function related to chronic obstructive pulmonary disease exacerbations.

Clinical considerations in individuals with α1-antitrypsin PI*SZ genotype.

α1-Antitrypsin deficiency (AATD), characterised by reduced levels or functionality of α1-antitrypsin (AAT), is a significantly underdiagnosed genetic condition that predisposes individuals to lung and liver disease. Most of the available data on AATD are based on the most common, severe deficiency genotype (PI*ZZ); therefore, treatment and monitoring requirements for individuals with the PI*SZ genotype, which is associated with a less severe AATD, are not as clear. Recent genetic data suggest the PI*SZ genotype may be significantly more prevalent than currently thought, due in part to less frequent identification in the clinic and less frequent reporting in registries. Intravenous AAT therapy, the only specific treatment for patients with AATD, has been shown to slow disease progression in PI*ZZ individuals; however, there is no specific evidence for AAT therapy in PI*SZ individuals, and it remains unclear whether AAT therapy should be considered in these patients. This narrative review evaluates the available data on the PI*SZ genotype, including genetic prevalence, the age of diagnosis and development of respiratory symptoms compared with PI*ZZ individuals, and the impact of factors such as index versus non-index identification and smoking history. In addition, the relevance of the putative 11 µM "protective threshold" for AAT therapy and the risk of liver disease in PI*SZ individuals is explored. The purpose of this review is to identify open research questions in this area, with the aim of optimising the future identification and management of PI*SZ individuals.

The Biological Effects of Double-Dose Alpha-1 Antitrypsin Augmentation Therapy. A Pilot Clinical Trial.

Rationale: Augmentation therapy with intravenous AAT (alpha-1 antitrypsin) is the only specific therapy for individuals with pulmonary disease from AAT deficiency (AATD). The recommended standard dose (SD; 60 mg/kg/wk) elevates AAT trough serum levels to around 50% of normal; however, outside of slowing emphysema progression, its effects in other clinical outcomes have not been rigorously proven.Objectives: To evaluate the biological effects of normalizing AAT trough levels with double-dose (DD) therapy (120 mg/kg/wk) in subjects with AATD already receiving SD therapy.Methods: Clinically stable subjects were evaluated after 4 weeks of SD therapy, followed by 4 weeks of DD therapy, and 4 weeks after return to SD therapy. At the end of each phase, BAL fluid (BALF) and plasma samples were obtained.Measurements and Main Results: DD therapy increased trough AAT levels to normal and, compared with SD therapy, reduced serine protease activity in BALF (elastase and cathepsin G), plasma elastase footprint (Aα-Val360), and markers of elastin degradation (desmosine/isodesmosine) in BALF. DD therapy also further downregulated BALF ILs and cytokines including Jak-STAT (Janus kinases-signal transducer and activator of transcription proteins), TNFα (tumor necrosis factor-α), and T-cell receptor signaling pathways, cytokines involved in macrophage migration, eosinophil recruitment, humoral and adaptive immunity, neutrophil activation, and cachexia. On restarting SD after DD treatment, a possible carryover effect was seen for several biological markers.Conclusions: Subjects with AATD on SD augmentation therapy still exhibit inflammation, protease activity, and elastin degradation that can be further improved by normalizing AAT levels. Higher AAT dosing than currently recommended may lead to enhanced clinical benefits and should be explored further.Clinical trial registered with www.clinicaltrials.gov (NCT01669421).

Free Desmosine is a Sensitive Marker of Smoke-Induced Emphysema.

PURPOSE: While the elastin-specific crosslinks, desmosine and isodesmosine (DID), are increased in blood, urine, and sputum of patients with clinically documented pulmonary emphysema, the usefulness of DID in detecting early lung injury remains untested. To this end, our laboratory has measured DID in a hamster model of smoke-induced emphysema, involving only minimal alveolar wall damage. METHODS: Animals were either treated with cigarette smoke for 2 h/day, 5 days/week, or exposed only to room air (controls) for a period of 3 months. DID levels in bronchoalveolar lavage fluid (BALF) and whole lungs were determined at monthly intervals, using liquid chromatography and tandem mass spectrometry. Lung surface area was also determined, as a measure of airspace enlargement. RESULTS: The portion of BALF DID not bound to peptides (free DID) was significantly higher in smoke-exposed animals at 2 months (9.2 vs 4.4 pg/mg protein; p < 0.05), whereas total BALF DID showed no significant increases over the course of the study, and total lung DID remained unchanged. There was a mild, but significant, loss of lung surface area in the smoke-exposed group at 2 months (28.8% vs 25.2%, p < 0.05), which showed no further progression, consistent with the return of free DID to control levels at 3 months. CONCLUSIONS: These findings support the hypothesis that free DID are sensitive indicators of smoke-induced lung injury. Measurement of free DID in smokers with minimally decreased lung mass may help determine the utility of this parameter as a test for incipient pulmonary emphysema.

Desmosine and Isodesmosine as a Novel Biomarker for Pulmonary Arterial Hypertension: A Pilot Study.

Delayed diagnosis is common in patients with pulmonary arterial hypertension (PAH). Right-sided heart catheterization, the gold standard for diagnosis, is invasive and cannot be applied for routine screening. Some biomarkers have been looked into; however, due to the lack of a clear pathological mechanism linking the marker to PAH, the search for an ideal one is still ongoing. Elastin is a significant structural constituent of blood vessels. Its synthesis involves cross-linking of monomers by 2 amino acids, desmosine and isodesmosine (D&I). Being extremely stable, elastin undergoes little metabolic turnover in healthy individuals resulting in very low levels of D&I amino acids in the human plasma, urine, or sputum. We hypothesized that in PAH patients, the elastin turnover is high; which in turn should result in elevated levels of D&I in plasma and urine. Using mass spectrometry, plasma and urine levels of D&I were measured in 20 consecutive patients with PAH confirmed by cardiac catheterization. The levels were compared with 13 healthy controls. The mean level of total plasma D&I in patients with PAH was 0.47 ng/mL and in controls was 0.19 ng/mL (P = 0.001). The mean levels of total D&I in the urine of PAH patients was 20.55 mg/g creatinine and in controls was 12.78 mg/g creatinine (P = 0.005). The mean level of free D&I in the urine of PAH patients was 10.34 mg/g creatinine and in controls was 2.52 mg/g creatinine (P < 0.001). This is the first study highlighting that the serum and urine D&I has a potential to be a novel screening biomarker for patients with PAH. It paves the way for larger studies to analyze its role in assessing for disease severity and response to treatment.

The Pattern of Elastic Fiber Breakdown in Bleomycin-Induced Pulmonary Fibrosis May Reflect Microarchitectural Changes.

INTRODUCTION: Desmosine and isodesmosine (DID) are unique elastin crosslinks that may serve as biomarkers for elastic fiber degradation in chronic obstructive pulmonary disease. Previously, our laboratory found that the ratio of free to peptide-bound DID in bronchoalveolar lavage fluid (BALF) showed a significant positive correlation with the extent of airspace enlargement in an elastase model of pulmonary emphysema. To further evaluate this hypothesis, our laboratory measured this ratio in a bleomycin (BLM) model of pulmonary fibrosis, which involved different microarchitectural changes than those associated with pulmonary emphysema. METHODS: Syrian hamsters were instilled intratracheally with 1.0 unit BLM in 0.2 ml of normal saline (controls received the vehicle alone), and BALF was analyzed for both free and total DID, using a combination of liquid chromatography and tandem mass spectrometry. RESULTS: Total BALF DID was significantly increased in hamsters receiving BLM at 1 week post-treatment (92 vs 13 pg/ml; p < 0.001), consistent with elastic fiber degradation. However, in contrast to elastase-induced emphysema, free/bound DID was lower in BLM-treated animals compared to controls at both 1 week (0.76 vs 0.84) and 2 weeks post-treatment (0.69 vs 0.86), though the differences were not statistically significant. CONCLUSIONS: These results indicate that it may be possible to identify specific pulmonary microarchitecture changes, based on the ratio of free to peptide-bound DID. It is speculated that the proportionate decrease in free DID in BLM-induced fibrosis may be due to preservation of intact elastic fibers as the lung injury progresses.

The Ratio of Free to Bound Desmosine and Isodesmosine May Reflect Emphysematous Changes in COPD.

BACKGROUND: The unique elastin crosslinks, desmosine and isodesmosine (DID) are significantly elevated in blood, urine, and sputum from patients with COPD, and may decline following treatment of the disease. However, the large degree of variance in this biomarker among COPD patients with similar levels of disease suggests that it has limited prognostic value with regard to the degree of lung disease in a given individual. As an alternative to measuring the total amount of DID, we propose using the ratio of free to peptide-bound DID, which may provide a better indication of overall lung disease. METHODS: To test this hypothesis, the free/bound DID ratio was measured in bronchoalveolar lavage fluid (BALF) from both hamsters with elastase-induced emphysema and controls not given the enzyme, using a combination of liquid chromatography and tandem mass spectroscopy. This ratio was then correlated with airspace enlargement, as measured by the mean percentage of lung surface area at ×100 microscopic magnification. RESULTS: There was a significant negative correlation between the free/bound DID ratio in BALF and lung surface area. However, there was no correlation between this ratio and total BALF DID, suggesting that free/bound DID is unrelated to the immediate rate of breakdown of elastic fibers, and may instead measure the cumulative effect of elastase injury in the lung. CONCLUSIONS: The free/bound DID ratio may be a useful measure of emphysematous changes in the lung and might also serve as a screening procedure for healthy smokers and other individuals at risk for developing COPD.

Synthesis and LC-MS/MS analysis of desmosine-CH2, a potential internal standard for the degraded elastin biomarker desmosine.

Desmosine-CH2, an analog of the elastic tissue degradation biomarker desmosine, can be regarded as a potential internal standard for precise quantification of desmosines by LC-MS/MS. In this study, the chemical synthesis of desmosine-CH2 was completed in 22% overall yield in five steps. The LC-MS/MS analysis of desmosine-CH2 was also achieved.

Stable deuterium internal standard for the isotope-dilution LC-MS/MS analysis of elastin degradation.

Chemical synthesis of the deuterium isotope desmosine-d4 has been achieved. This isotopic compound possesses all four deuterium atoms at the alkanyl carbons of the alkyl amino acid substitution in the desmosine molecule and is stable toward acid hydrolysis; this is required in the measurement of two crosslinking molecules, desmosine and isodesmosine, as biomarkers of elastic tissue degradation. The degradation of elastin occurs in several widely prevalent diseases. The synthesized desmosine-d4 is used as the internal standard to develop an accurate and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry analysis, which can serve as a generalized method for an accurate analysis of desmosine and isodesmosine as biomarkers in many types of biological tissues involving elastin degradation.

Neutrophil elastase-mediated lung disease.

Elastases of both the neutrophil and macrophage have been implicated in lung disease initiation and progression. Although it is unlikely that these proteases evolved for the purpose of injuring lung tissue, the elastin-rich connective tissue framework of the lungs appears to be particularly susceptible to the action of elastolytic proteases. Assuming that neutrophil elastase most likely plays a role in the migration of neutrophils toward a site of inflammation and degradation of proteins from invading organisms or other products of the inflammatory response, it is the role of inhibitors of this protease to protect normal tissues from its effects. In alpha-1 antitrypsin deficiency we find an experiment of nature that disrupts this protease-anti-protease balance, resulting in an increased risk of destructive lung disease.

Alpha-1 antitrypsin augmentation therapy and biomarkers of elastin degradation.

BACKGROUND: Intravenous alpha-1 antitrypsin protein (AAT) augmentation is a prescribed therapy for severe, genetically determined, alpha-1 antitrypsin deficiency (AATD), a genetic basis for pulmonary emphysema. AAT, a predominant systemic inhibitor of neutrophil elastase thus far has not been shown to decrease elastin degradation in a significant number of patients on this therapy. The objective of this study was to compare levels of biomarkers of elastin degradation in plasma, bronchoalveolar lavage (BALF) fluid and urine before and after beginning AAT augmentation therapy in patients with AATD. METHODS: Desmosine and isodesmosine (DI), which occur only in elastin, are amino acid cross-links in mature elastin. Levels of DI in body fluids measure degradation of elastin and can be measured more specifically by mass spectrometry. This method was used to measure DI levels in plasma, bronchoalveolar lavage fluid and urine in cohorts of severe AATD patients on augmentation, not on augmentation and before and after the initiation of augmentation therapy. RESULTS: Statistically significant reductions in plasma DI and in BALF DI were demonstrated in AATD patients receiving intravenous (IV) augmentation therapy as compared with those not receiving it. Administration by aerosol also produced statistically significant reductions in levels of DI in BALF. CONCLUSIONS: Results indicate that the currently prescribed doses of AAT augmentation inhibit neutrophil elastase adequately to reduce elastin degradation, both systemically and in the lung per se. The currently prescribed doses did not reduce elastin degradation to control levels, which may be possible with higher doses.

Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency.

BACKGROUND: The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency. METHODS: The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV1 percent of predicted and FEV1/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD. RESULTS: Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV1 percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV1 percent of predicted and pre-bronchodilator FEV1/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed. CONCLUSIONS: IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.

Matrix elastin: a promising biomarker for chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major health problem worldwide and is now the third leading cause of death in the United States. There is a lack of therapies that can stop progression of the disease and improve survival. New drug discovery can be aided by the development of biomarkers, which can act as indicators of severity in the course of the disease and responses to therapy. This perspective brings together the laboratory and clinical evidence, which suggest that elastin degradation products can fulfill the need for such a biomarker. Elastin is a recognized target for injury in COPD. The amino acids desmosine and isodesmosine exist only in matrix elastin; can be measured specifically and sensitively in plasma, urine, and sputum; and indicate changes in the systemic balance between elastase activity and elastase inhibition brought on by the systemic inflammatory state. The biomarker levels in sputum reflect the state of elastin degradation in the lung specifically. Clinical data accumulated over several decades indicate correlations of desmosine and isodesmosine levels with COPD of varying severity and responses to therapy.

Elastin Degradation and Lung Function Deterioration with Remote Secondhand Tobacco Smoke Exposure in Never-smokers.

Background: Prolonged past exposure to secondhand tobacco smoke (SHS) in never-smokers is associated with abnormal lung function and reduced diffusing capacity suggestive of an associated lung tissue injury and damage. The mechanisms by which past SHS exposure may contribute to lung tissue damage are unknown. Elastin is a major constituent of extracellular matrix in lung parenchyma. Objective: To determine whether past exposure to SHS is associated with ongoing lung tissue damage as indicated by elevated elastin degradation products that are linked to lung function. Methods: We measured the plasma levels of elastin degradation markers (EDM) from 193 never-smoking flight attendants with a history of remote SHS exposure in aircraft cabins and 103 nonsmoking flight attendants or sea-level control participants without such history of cabin SHS exposure and examined those levels versus their lung function with adjustment for covariates. The cabin SHS exposure was estimated based on airline employment history and years of the smoking ban enactment. Results: The median [interquartile range] plasma EDM level for all participants was 0.30 [0.24-0.36] ng/mL with a total range of 0.16-0.65 ng/mL. Plasma EDM levels were elevated in those with a history of exposure to cabin SHS compared to those not exposed (0.33±0.08 versus 0.26±0.06 ng/mL; age- and sex-adjusted P<0.001). In those with a history of cabin SHS exposure, higher EDM levels were associated with a lower diffusing capacity (parameter estimate [PE] 95% [confidence interval(CI)]=4.2 [0.4-8.0] %predicted decrease per 0.1 ng/mL increase in EDM; P=0.030). Furthermore, EDM levels were inversely associated with forced expiratory volume in 1 second (FEV1), FEV1 to forced vital capacity (FVC) ratio , and forced expiratory flow rate between 25% and 75% ( FEF25%-75%) (PE [95%CI]=5.8 [2.1-9.4], 4.0 [2.2-5.7], and 12.5 [5.8-19.2] %predicted decrease per 0.1 ng/mL increase in EDM, respectively; P<0.001). Plasma EDM mediated a substantial fraction of the association of SHS with FEV1, FVC, and FEF25%-75% (P<0.05). Conclusions: Long after past exposure to SHS, there is ongoing elastin degradation beyond what is expected from the aging process, which likely contributes to lower lung function and a reduced pulmonary capillary bed as seen in chronic obstructive pulmonary disease (COPD).

Therapeutic effects of hyaluronan on smoke-induced elastic fiber injury: does delayed treatment affect efficacy?

Aerosolized hyaluronan (HA) has been previously shown to prevent cigarette smoke-induced airspace enlargement and elastic fiber injury in mice when given concurrently with smoke. In the present study, a more stringent test of the therapeutic potential of HA was performed by delaying treatment with this agent for 1 month. After treatment with cigarette smoke for 3 h per day for 5 days per week for 1 month, mice (DBA/2J) began receiving aerosolized HA (0.1%) for 1 h prior to smoke exposure (controls were given aerosolized water). The results indicate that much of the damage to the lung elastic fibers occurred within the first several months of smoke exposure, as measured by levels of desmosine and isodesmosine (DID) in bronchoalveolar lavage fluid (BALF). In contrast to previously published studies, where concurrent administration of aerosolized HA significantly reduced BALF DID levels within 3 months of smoke exposure, the same effect was not seen until 6 months when HA treatment was delayed. However, despite the prolonged breakdown of elastic fibers in the current study, a significant reduction in airspace enlargement was observed after only 2 months of HA treatment. These findings provide further support for testing this agent in patients with pre-existing chronic obstructive pulmonary disease.

A 28-day clinical trial of aerosolized hyaluronan in alpha-1 antiprotease deficiency COPD using desmosine as a surrogate marker for drug efficacy.

INTRODUCTION: A previous 2-week clinical trial of aerosolized hyaluronan (HA) in COPD showed a rapid reduction in lung elastic fiber breakdown, as measured by sputum levels of the unique elastin crosslinks, desmosine and isodesmosine (DID). To further assess the therapeutic efficacy of HA and the utility of DID as surrogate markers for the development of pulmonary emphysema, we have conducted a 28-day randomized, double-blind, placebo-controlled, phase 2 trial of HA involving 27 subjects with alpha-1 antiprotease deficiency COPD. METHODS: The study drug consisted of a 3 ml inhalation solution containing 0.03% HA with an average molecular weight of 150 kDa that was self-administered twice daily. DID levels were measured in urine, sputum, and plasma using tandem mass spectrometry. RESULTS: Free urine DID in the HA group showed a significant negative correlation with time between days 14 and 35 (r = -1.0, p = 0.023) and was statistically significantly decreased from baseline at day 35 (15.4 vs 14.2 ng/mg creatinine, p = 0.035). A marked decrease in sputum DID was also seen in the HA group between days 1 and 28 (0.96 vs 0.18 ng/mg protein), but the difference was not significant, possibly due to the small number of adequate specimens. Plasma DID remained unchanged following HA treatment and no significant reductions in urine, sputum, or plasma DID were seen in the placebo group. CONCLUSIONS: The results support additional clinical trials to further evaluate the therapeutic effect of HA and the use of DID as a real-time marker of drug efficacy.

Characterization of peptide fragments from lung elastin degradation in chronic obstructive pulmonary disease.

This study presents a method for detecting and characterizing peptides of elastin that result from lung matrix injury in chronic obstructive pulmonary disease (COPD). Lung elastin degradation was studied by two representative in vivo elastases, human neutrophil elastase (HNE) and macrophage metalloproteinase (MMP12). The resulting peptide mixtures were analyzed by high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MSMS) to characterize 40 elastin-derived peptides (EDPs), 24 from HNE and 16 from MMP12 digestions. The peptides constitute major EDPs that are solubilized by the enzymatic digestion. Using the selected reaction monitoring (SRM) from LC/MSMS analysis, the transition ions of the peptides were used to investigate the presence of the peptides in selected body fluids of chronic obstructive pulmonary disease (COPD) patients. Four peptides, GYPI, APGVGV, GLGAFPA, and VGVLPGVPT, were detected in plasma or sputum of some COPD patients but not in normal controls. A hexapeptide VGVAPG, which had been widely studied for its chemotactic activity for a possible pathogenic role in COPD, was not detected in lung EDPs by HNE or MMP12 digestion, but only by porcine pancreatic elastase (PPE) digestion. This study demonstrates a practical methodology to study peptides from matrix degradations in pulmonary disease and a means of investigating their pathogenesis.